1. Field of the Invention
The present invention relates to glyoxylation-stabilized, arginine-containing synthetic lytic peptide compositions with enhanced resistance to proteolytic digestion, and to methods of making the same.
2. Description of Related Art
Naturally occurring amphipathic lytic peptides play an important if not critical role as immunological agents in insects and have some, albeit secondary, defense functions in a range of other animals. The function of these peptides is to destroy prokaryotic and other non-host cells by disrupting the cell membrane and promoting cell lysis. Common features of these naturally occurring amphipathic, lytic peptides include an overall basic charge, a small size (23-39 amino acid residues), and the ability to form amphipathic .alpha.-helices. Several types of amphipathic lytic peptides have been identified: cecropins (described in U.S. Pat. Nos. 4,355,104 and 4,520,016 to Hultmark et al.), defensins, sarcotoxins, melittin, and magainins (described in U.S. Pat. No. 4,810,777 to Zasloff). Each of these peptide types is distinguished by sequence and secondary structure characteristics.
Several hypotheses have been suggested for the mechanism of action of the lytic peptides: disruption of the membrane lipid bilayer by the amphipathic .alpha.-helix portion of the lytic peptide; lytic peptide formation of ion channels, which results in osmotically induced cytolysis; lytic peptide promotion of protein aggregation, which results in ion channel formation; and lytic peptide-induced release of phospholipids. Whatever the mechanism of lytic peptide-induced membrane damage, an ordered secondary conformation such as an .alpha.-amphipathic helix and positive charge density are features that appear to participate in the function of the lytic peptides.
Active analogs of naturally occurring lytic peptides have been produced and tested in vitro against a variety of prokaryotic and eukaryotic cell types (see for example Arrowood, M. J., et al. J. Protozool. 38: 161s [1991]; Jaynes, J. M., et al. FASEB J. 2: 2878 [1988]), including: gram positive and gram negative bacteria, fungi, yeast, envelope viruses, virus-infected eukaryotic cells, and neoplastic or transformed mammalian cells. The results from these studies indicate that many of the synthetic lytic peptide analogs have similar or higher levels of lytic activity for many different types of cells, compared to the naturally occurring forms. In addition, the peptide concentration required to lyse microbial pathogens such as-protozoans, yeast, and bacteria does not lyse normal mammalian cells.
The specificity of the lytic action depends upon the sequence and structure of the peptide, the concentration of the peptide, and the type of membrane with which it interacts. Jaynes et aL Peptide Research 2: 157 (1989) discuss the altered cytoskeletal characteristics of transformed or neoplastic mammalian cells that make them susceptible to lysis by the peptides. In these experiments, normal, human non-transformed cells remained unaffected at a given peptide concentration while transformed cells were lysed; however, when normal cells were treated with the cytoskeletal inhibitors cytochalasin D or colchicine, sensitivity to lysis increased. The experiments show that the action of lytic peptides on normal mammalian cells is limited. This resistance to lysis was most probably due to the well-developed cytoskeletal network of normal cells. In contrast, transformed cell lines which have well-known cytoskeletal deficiencies were sensitive to lysis. Because of differences in cellular sensitivity to lysis, amphipathic peptide concentration can be manipulated to effect lysis of one cell type but not another at the same locus.
Synthetic lytic peptide analogs can also act as agents of eukaryotic cell proliferation. Peptides that promote lysis of transformed cells will, at lower concentrations. promote cell proliferation in some cell types. This stimulatory activity is thought to depend on the channel-forming capability of the peptides, which somehow stimulates nutrient uptake, calcium influx or metabolite release, thereby stimulating cell proliferation (see Jaynes, J. M. Drug News & Perspectives 3: 69 [1990]; and Reed, W. A. et al. Molecular Reproduction and Development 31: 106 [1992]). Thus, at a given concentration, these peptides stimulate or create channels that can be beneficial to the normal mammalian cell in a benign environment where it is not important to exclude toxic compounds.
The synthetic lytic peptide analogs typically contain as few as 12 and as many as 40 amino acid residues. A phenylalanine residue is often positioned at the amino terminus of the protein to provide an aromatic moiety analogous to the tryptophan residue located near the amino terminus of natural cecropins and a UV-absorbing moiety with which to monitor the purification of the synthetic peptide. The basis for the design of these lytic peptide analogs is that an amphipathic peptide of minimal length and containing overall positive charge density effects lytic activity. Peptides that have the structural motif of a .beta.-pleated sheet and overall positive charge density can also effect lytic activity.
As discussed in the preceding paragraph, in vitro laboratory tests of the lytic peptide analogs have been successful. However, the use of the lytic peptide analogs in vivo could be considerably limited in circumstances where proteases may digest the peptide analogs before sufficient pathogen cell lysis has occurred. In particular, the high concentration of positively charged amino acids such as lysine and arginine make the synthetic peptides susceptible to tryptic digestion. The secondary conformation of the peptides sequesters the hydrophobic amino acid residues, thus shielding them from interaction with proteases such as chymotrypsin, which hydrolyzes peptides at bulky or aromatic amino acid residues. This proteolytic susceptibility is a general problem for peptides and proteins when used in vivo. Many techniques are suitable for stabilizing proteins for in vitro use but are not appropriate for in vivo or oral administration to humans and animals.
Several studies teach that chemical modification of arginine residues has been used to study structure-function relationships in a variety of naturally occurring proteins and their substrates. For example, Busby et al., Arch. Biochem. Biophys 177: 552 (1976) teach that chemical modification of arginine residues with 1,2-cyclohexanedione can be used to study the essential nature of arginine residues in the maintenance of biological activity. This report states that the arginine residues in the test protein, plasma .alpha.-1 anti-trypsin, do not participate in the catalytic reaction.
Lin et al., Biochim. et Biophys. Acta 1159: 255 (1992) teach that .alpha.-bungarotoxin, a long-chain neurotoxin containing three arginine residues, can be derivatized on the arginine residues using either 1,2-cyclohexanedione or p-hydroxylphenylglyoxal. The effect of arginine modification on biological activity was tested by examining the binding activity of the modified toxin to the nicotinic acetylcholine receptor. None of the modified arginine residues were essential for binding to the receptor, however, the modification resulted in diminution of lethality and binding affinity. Thus, the article relates only to binding characteristics and biological activity of a modified neurotoxin and does not address enhanced proteolytic resistance of the protein.
Patthy et al., J. Biol. Chem. 250: 557 (1975) and J. Biol. Chem. 250: 565 (1975) teach that modification of arginine residues in lysozyme using 2,3-butanedione, phenylglyoxal or glyoxal showed that lysozyme retained biological activity.
Accordingly, it would be a significant advance in the art to provide a method of producing chemically modified physiologically active lytic peptides that have enhanced resistance to proteolysis.
It would be particularly desirable to provide a method of producing such peptides so that the modified peptides have enhanced proteolytic stability and retain their physiological activity for in viva applications against pathogenic microbial organisms such as bacteria, yeast, fungi, and protozoans; neoplastic or transformed cells; envelope viruses; and virally-infected cells.
These and other objects and advantages will be more fully apparent from the ensuing disclosure and claims.